Team:Freiburg/parts/improvement

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Improvement of a Registry part

We decided to improve the BioBrick BBa_K128001 coding for the HA-tag – a tag which is commonly used to detect proteins. As it is cloned to all of our designed devices, we were in need of a well-functioning and easy-clonable HA-part.

The BBa_K128001 contains the RFC23 pre- and suffix (silver standard) which can be used for the construction of in-frame-fusion parts. In contrast to the RFC25 biobrick (which is also suitable for functional part fusions) there is no start codon and stop codon automatically included in the prefix and suffix.

That’s why we designed a new version of the HA-tag BBa_K128001 with an identical sequence of amino acids but with an RFC25 pre- and suffix. This has following advantages:

• Cloning the HA-tag BBa_K1150016 N-terminal to other fusion parts leads to an automatic transcriptional start due to the introduced ATG in the RFC25 prefix.
• Cloning the HA-tag BBa_K1150016 C-terminal to other fusion parts leads to an automatic transcriptional stop due to the introduced stop codon in the RFC25 suffix.

As tags are often fused at the end (C- or N-terminal) of proteins, this HA-tag facilitates cloning.

Fig.1: BBa_K1150016 with RFC25 prefix and suffix.

Before bringing this tag into the iGEM-standard we first tested its functionality via Western blot detection (Fig.2) . As it worked fine in mammalian cells, we used this sequence for our standardized constructs. Testing of our standardized devices including the BBa_K1150016 worked well and also western blot detection was possible (Fig.3) .

Fig.2: HA-tag fused to CRY2-C-RAF-CIBN-protein encoded on the pEF6/V5-His-TOPO plasmid.

Fig.3: HA-tag fused to different dcas9-fusion proteins encoded on the RFC25 pSB1C3 backbone.